To determine the gene
expression profile of aging in rockfish, total RNA was isolated from two pools of rockfish
liver samples: twelve year olds and older rockfish in their thirties. Sufficient numbers
of samples were available to isolate RNA from two groups in each age range to allow for
biological replication of results. RNA was labeled using standard amino-allyl labeling
protocols, and hybridized overnight to an array with oligonucleotides spotted in duplicate
in separate quadrants across a glass slide. A long-oligonucleotide (65mers) zebrafish
array set was obtained from Compugen, Inc. (http://www.labonweb.com/),
which contains 16,399 oligonucleotides, representing 16,228 LEADSTM clusters (likely
genes) plus 171 control oligonucleotides.

Because standard protocols generate lower signal intensities when applied to long
oligonucleotide spotted arrays, several modifications to the standard labeling and
hybridization protocols have been developed to improve signal-to-noise and overall signal
intensity on the long oligonucleotide platform. Our expectation is that the high degree of
sequence conservation among fish species will enable hybridization of rockfish RNA with
the zebrafish sequences that are commercially available. The homology between zebrafish
and rockfish is perhaps 60% (an estimate by Phylonix, a contract research organization
providing in vivo zebrafish assays). Hybridization conditions will be relaxed should
signal intensities be insufficient. With the large number of genes present on the
oligonucleotide array, we expect to obtain a large amount of information regarding changes
in the gene expression profile in rockfish liver during aging. These changes will be
compared with expression data from shorter lived species to identify genes that may be
responsible for extreme longevity in rockfish.

The first experiment with the rockfish RNA did not produce results. The controls
worked, but there were no signals from the rockfish RNA. The experiment is being repeated
under different conditions that may increase the signal, with new RNA being prepped for
the next experiment.

Caption: Zebrafish Micro-array fluorescence output shown, following hybridization
of fluorescently labeled zebrafish RNA to robotically spotted oligonucleotides affixed to
a glass slide. This technique was used for the rockfish liver samples.

Each "side" of the image is from a microscope slide that a "robot"
has dotted with DNA. Each spot is a different sequence of DNA. When the RNA is extracted
from young and from old rockfish, it is fluorescently labeled and individual genes attach
(hybridize) to the spot that matches their sequence. There are over 16,000 spots and
therefore genes on the slides. Since the RNA is labeled with a fluorescent dye, the
fluorescences from each spot corresponds to the amount of each gene that is present in the
RNA sample. By comparing one slide from the young and one slide from the old, the
difference between the two for each gene can be calculated. Therefore we can identify
which genes get turned on or up with aging, and which genes get turned down or off.

Recent literature has pointed out that at least some gene mutations that extend
lifespan are in genes that involve increased resistance to oxidative stress, thermal
stress and ultraviolet radiation. Increased lifespan could depend on the organisms ability
to fend off insult by increased constitutive levels of protein products of some or all of
these protective genes. Hsp70 (heat shock protein 70) and hsc70 (heat shock cognate 70)
statement has been shown to play a role in the cascade of events dealing with protection
from oxidative, thermal and ultraviolet insult. Prolonged exposure to innumerable
stressors can result in cell death. The presence of these and other molecular chaperones
may become extremely important in understanding how non-dividing cells survive a longer
lifespan.

This study looked at the gene statement of the heat shock 70 family in rockfish. We
compared constitutive levels in long-lived species of rockfish with those of other short-
and long-lived organisms already examined to determine whether hsp70 levels change with
age. We used western blot analysis, with antibodies available against a region that is vey
homologus across species, and has been used in some of the published fish studies thus
far.

The research results on rockfish liver samples ranging from 12 to 77 years old showed
very low levels of hsp70 as compared to prior tests performed on mice. This would appear
to eliminate heat shock protein as a factor in rockfish longevity.

(3 - January 2002) SOD antioxidant defense changes with age.

Glenn S. Gerhard, Weis
Center for Research, Danville, PA.

Reactive oxygen species (ROS), such as superoxide anion and hydrogen peroxide, are
generated in aerobic organisms through normal metabolism. ROS are involved in normal
processes such as intracellular signaling and defense against micro-organisms. In
addition, however, ROS cause oxidative damage, which may be involved in the aging process.
Lipid peroxidation, DNA damage, and protein oxidation mediated by ROS have been found to
increase during aging in various tissues of many organisms.

Some long-lived rockfish such as Yelloweye rockfish (Sebastes
ruberrimus), appear to exhibit negligible senescence (absence of a significant increase in
mortality rate with age). It is therefore of interest to determine whether processes
relating to oxidative damage change with age in these animals. A wide array of enzymatic
antioxidant defenses exist to counteract ROS, including superoxide dismutase (SOD) and
catalase (CAT). SOD converts the superoxide radical to the less reactive H2O2. MnSOD is a
nuclear-encoded enzyme that is located within the mitochondria. CuZnSOD may play a major
role in detoxifying superoxide anion radicals located in the cytoplasm. We determined
whether levels of these enzymes showed changes with age in negligibly-senescing rockfish.

Protein levels of MnSOD and CuZnSOD in liver samples obtained from rockfish were
quantified by Western blot (first figure) and plotted versus age (second figure). A
discordant relationship with age appears to exist for the two enzymes: MnSOD fell with age
while CuZnSOD increased with the age of the rockfish. The increase with age in CuZnSOD is
an intriguing finding given the powerful antioxidant status of SOD.

This study is investigating electron transport abnormalities and mitochondrial DNA
mutations in rockfish heart tissue ranging in age from 12 to 83 years old. Judd has
successfully amplified a portion of the cytochrome b gene. This was critical as his
initial experimental design involved using the cytochrome b gene as the starting point for
full genome PCR amplification.

With the recent publication of the complete mitochondrial genome for a closely related
species, Sebastes schlegeli, he has revised his experimental protocol. New primer sets,
based on the S. schlegeli and the limited sequence data for S. ruberrimus, have been
designed that will allow him to amplify the major arc of the mitochondrial genome.
Currently those primers are being used to investigate abnormalities and mutations.

In a joint collaboration, the project's turtle ecologist will use the Kronos Science
Complete Assessment tests (shown in chart for Project 8 - Rockfish Biochemical Profiling).
Turtles have been marked for many decades at several reserves in the United States
(Congdon 1996). At the E.S. George Reserve in Michigan, some Blanding's turtles were
marked as adults in the 1950's and remain in the population today; see picture below.
Blood serum will be drawn from these and younger turtles. The measurements will identify a
set of biochemical markers of age, which can then be compared with rockfish and other
animals. Justin attempted to trap several turtles as part of an ecology assessment in 2001
but was unsuccessful in collecting samples. He has subsequently applied for general
funding with which he can collect and analyze blood serum samples for this project.

Tory received 16 rockfish liver samples ranging in age from teen-age years to over 90
years old. He measured DNA damage in both mitochondrial and nuclear DNA (Helbock et al.
for protocol). Since up to 95% of free-radical production occurs in the mitochondria, this
information from a long-lived species could be very revealing. The preliminary evidence
suggests there is no age-related increase in steady state 8-oxo-deoxyguanosine among the
different aged samples (shown is table comparing 8-oxo-dG damage). The numbers, however,
were several times higher than seen in rat liver tissue, so a larger sample size is
necessary to verify if this is due to species differences or to artificial oxidation,
which would still tend to increase relative differences uniformly.

Berislav received rockfish heart and brain samples up to 80+ years old. He has applied
for funding to construct a complementary DNA (cDNA) library, using subtractive
hybridization, a relatively expensive process. When funded he will identify active genes
in the younger and older rockfish. This information can then be compared with
shorter-lived rockfish.

(8 - October 2000) Rockfish Biochemical profiling.

Gregor Cailliet, Moss Landing Marine
Laboratories, Moss Landing, CA.

Greg collected blood serum samples from several long-lived rockfish. This collaboration
with Kronos Science was intended to run comprehensive biochemical profiling tests (the
same biochemical tests as for turtles above), and to eventually collect and compare the
results with short-lived rockfish. This would have identified possibly over a hundred
biochemical markers of age, focusing on cardiovascular function, oxidative stress
(including CoQ10 and ORAC tests), and a profile of vitamins, minerals and hormones.
However, the blood preparation could not be prepared to the specifications for the Kronos
tests, which are normally run with human blood serum. Without funding the project was
unable to continue collecting samples to get usable research results.